ASSEMBLY AND ACTIVATION OF ENZYME-SSDNA COMPLEXES

这项研究计划的主要目标是阐明 单链DNA上酶复合物的组装机制 并激活。特异性酶与ssDNA的相互作用是一种 DNA复制机制的基本特征。重组和 所有活细胞都能进行的修复。组装和激活 酶-ssDNA复合物是复杂的，然而，由于螺旋的存在， 不稳定蛋白（HDPs），紧密结合，合作， ssDNA为了与ssDNA结合并激活其催化活性， 必须首先取代HDPs，这是一个能量上不利的过程。因此 似乎需要特殊的HDP位移机构来加载 将酶转移到预先存在的HDP-ssDNA基质上。我们建议研究这些 位移和加载机制，通过研究分子步骤 参与了两种不同的模型酶的组装和激活- ssDNA复合物：DNA解旋酶的复合物，噬菌体T4基因41 蛋白（gp 41）和一般重组酶的T4 uvsX蛋白。 每种酶-ssDNA复合物的组装和激活似乎 需要尽可能多的蛋白质-ssDNA和/或蛋白质-蛋白质相互作用 作为三种不同的噬菌体蛋白：酶本身， 组装因子和特异性HDP-T4基因32蛋白（gp 32）。我们 因此，具体的目标是解剖蛋白质-ssDNA和蛋白质- 蛋白质相互作用参与gp 41和uvsX加载到gp 32- ssDNA这将通过分析两者的相互作用来实现 组装因子与ssDNA，与gp 32和与它们的酶伴侣。 蛋白质-ssDNA相互作用将通过荧光直接检查 光谱法和间接耦合分光光度测定法。 蛋白质- 蛋白质相互作用将通过结合蛋白质亲和力 色谱法和沉降法，并通过分离和 特异性改变的蛋白质的表征。预计在 这些研究将确定重要的大分子相互作用， 酶-ssDNA复合物的组装和活化所需的，和 用其他蛋白质取代ssDNA中的HDPs。以更 一般意义上，确定所需的大分子相互作用， ssDNA上一种蛋白质被另一种蛋白质置换应该有助于 我们对许多其他蛋白质置换反应的理解 在各种遗传过程中，包括 核小体通过转录因子和一种蛋白质的置换 由另一个在处理和细胞内运输的信使 RNA。

The broad goal of this research program is to elucidate the molecular mechanisms by which enzyme complexes on single-stranded DNA are assembled and activated. The interaction of specific enzymes with ssDNA is an essential feature of the mechanisms of DNA replication. recombination, and repair that occur in all living cells. The assembly and activation of enzyme-ssDNA complexes is complicated, however, by the presence of helix- destabilizing proteins (HDPs) that bind tightly and cooperatively to ssDNA. To bind to ssDNA and activate its catalytic activities, an enzyme must first displace HDPs, an energetically unfavorable process. Therefore special HDP displacement mechanisms appear to be required to load an enzyme onto a preexisting HDP-ssDNA matrix. We propose to study these displacement and loading mechanisms by investigating the molecular steps involved in the assembly and activation of two different model enzyme- ssDNA complexes: that of a DNA helicase, the bacteriophage T4 gene 41 protein (gp41), and that of a general recombinase, the T4 uvsX protein. The assembly and activation of each enzyme-ssDNA complex, appears to require the protein-ssDNA and/or protein-protein interactions of as many as three different phage proteins: the enzyme itself, an enzyme-specific assembly factor, and a specific HDP-- the T4 gene 32 protein (gp32). Our specific objective is therefore to dissect the protein-ssDNA and protein- protein interactions involved in the loading of gp41 and uvsX onto gp32- ssDNA. This will be accomplished by analyzing the interactions of the two assembly factors with ssDNA, with gp32 and with their enzyme partners. Protein-ssDNA interactions will be examined directly by fluorescence spectroscopy and indirectly by coupled spectrophotometric assay. Protein- protein interactions will be examined by a combination of protein affinity chromatography and sedimentation methods and by the isolation and characterization of specifically altered proteins. It is expected that these studies will identify the important macromolecular interactions required for the assembly and activation of enzyme-ssDNA complexes, and for the displacement of HDPs from ssDNA by other proteins. In a more general sense, identifying the macromolecular interactions required for the displacement of one protein by another on ssDNA should contribute to our understanding of many other protein displacement reactions that occur during the various genetic processes, including the displacement of nucleosomes by transcription factors and the displacement of one protein by another during the processing and intracellular transport of messenger RNAs.